Response of Oxidative Stress and Antioxidant System in Pea Plants Exposed to Drought and Boron Nanoparticles

Pea plants are sensitive to water shortages, making them less attractive to farmers. Hoping to reduce the adverse effects of drought on peas and considering the benefits of boron, this study aimed to investigate the impact of boron nanoparticles on the antioxidant system and oxidative stress biomarkers in drought-stressed peas. Experiments were performed in a greenhouse. Pea plants were treated with a suspension of B2O3 nanoparticles at 12.5, 25, and 50 ppm concentrations before ten days of water shortage. Drought effects were induced by maintaining 30% substrate moisture. This study investigated the properties of the nanoparticle suspension and different application methods for spraying and watering pea plants. The effects of B2O3 nanoparticles and drought were determined on pea growth indicators, oxidative stress biomarkers, and enzymatic and non-enzymatic antioxidants. Spraying with B2O3 nanoparticles at 12.5 ppm most effectively stimulated phenol accumulation; FRAP, DPPH, and ABTS antioxidant capacity; and APX, SOD, GPX, and CAT enzyme activity in pea leaves exposed to drought. In addition, B2O3 nanoparticles reduced the amount of MDA and H2O2 in pea plants grown on a substrate with insufficient moisture. The most substantial positive effect was found on peas affected by drought after spraying them with 12.5 ppm of B2O3 nanoparticles. B2O3 nanoparticles positively affected the pea height, leaf area, number of nodules, and yield.


Introduction
Nanotechnology is an advanced field of science, the use of which ranges from standard household chemicals and cosmetics to precision agriculture, the development of medicines and medical devices, and their use in space technology for effective shielding and energy storage. It is part of the future, but the environmental impact needs to be studied before its widespread use. Furthermore, considering climate change, the depletion of fossil minerals, and sustainable farming, it is essential to adopt new methods. This manuscript investigates drought and boron (B) nanoparticles' (NPs) effects on pea plants. It is known that B is necessary for plants of the leguminous family because it makes nitrogen fixation more efficient. In addition, B plays a crucial role in the formation and stability of cell walls, supports the functional and structural integrity of biological membranes, promotes the movement of sugar or energy to the growing parts of plants, and positively affects pollination and seed sets.
B is also known to stimulate both enzymatic and non-enzymatic antioxidant activity. Many scientific publications highlight the benefits of bulk B on the plant antioxidant system during different stress conditions [1-3], but only a few investigate the effect of B 2 O 3 NPs on plants [4][5][6].
Recent research has shown that the application of 150 mg L −1 arbuscular mycorrhiza (AM) with 100 mg L −1 B 2 O 3 NPs can significantly increase the height, the number of leaves,

Research Conditions
The experiments were carried out during the spring-summer periods of two years (2019-2020) in two greenhouses (3 × 6 m; h = 2 m) at the Lithuanian Research Centre for Agriculture 41 and Forestry, Institute of Horticulture, Babtai, Lithuania (55 • 05 08.4 N 23 • 48 03.5 E, at an altitude of 51 m; moderate climate zone of the northern hemisphere). Before sowing, green pea seeds were sterilized in 5% sodium hypochlorite (NaClO) solution for 15 min to ensure surface sterility [14] and rinsed gently with deionized water several times. Then, the seeds were soaked in water for 24 h. Ten seeds were sown in 10-L volume plastic pots (7 pots per treatment, arranged randomized) and filled with~8 kg of soil mixture (volume of 7:1 soil to perlite ratio, respectively). The soil was heavy loam with a particle size distribution, pH 7.4 ± 0.1; concentration of humus-3.6 ± 0.1%; P 2 O 5 -243 ± 8 mg kg −1 ; K 2 O-348 ± 37 mg kg −1 ; NH 4 -4 ± 0.6 mg kg −1 ; NO 3 -22 ± 0.9 mg kg −1 ; SiO 2 -39 ± 0.8 mg kg −1 ; B-0.02 ± 0.001 mg kg −1 (after the experiment, the composition of the soil was also analyzed). Pea seedlings were thinned to 7 plants per pot five days after sowing. After 16 days of cultivation, the peas were fertilized with 7 g pot −1 ammonium nitrate (NH 4 NO 3 , Merck KGaA, Darmstadt, Germany). The peas were sprayed with fungicides because the green pea cultivar 'Respect' is more susceptible to powdery mildew, even when grown in a greenhouse. Pots were irrigated with water by a graduated cylinder daily to 80% of substrate moisture (SM) using a substrate moisture sensor (Delta-T devices, HH2 moisture meter, Cambridge, United Kingdom) for 35 days. Plants were grown under a naturalday-length photoperiod. The average day/night temperature was 22.2/14.4 • C; relative air humidity-58/77 ± 5% before exposure; during the ten days of drought treatment, the average day/night temperature was 25.4/16.6 • C, and the relative air humidity was 53/75 ± 5%, and data were measured throughout the experiment (Termio+ data logger, Lubawka, Poland) in the first year. The conditions of the second experiment were as follows: the day/night average temperature was 24.2/14.4 • C; relative air humidity-54/75 ± 5% before exposure; during the ten days of drought exposure, the day/night Antioxidants 2023, 12, 528 3 of 15 average temperature was 26.2/17.0 • C, relative air humidity was 50/73 ± 5%. After the peas reached the 40 BBCH growth stage [15], they were foliar sprayed until full wetting (ca. 14 ± 0.5 mL plant −1 ) or watered (100 ± 1 mL per pot) with suspensions containing 12.5 ppm, 25 ppm, and 50 ppm concentrations of B 2 O 3 NPs; control (NP-untreated) were watered or sprayed with water. After the application of NPs, the watering of one part of the pea plants was stopped, and drought stress was initiated (30% SM). Substrate moisture was measured every day at the same time and maintained at 30% by watering when needed. In contrast, another part of the pea and control plants was irrigated with water to maintain regular soil moisture (80% SM) throughout the experiment. These regimes were applied for ten days until harvest. After each treatment, plants were harvested after reaching the BBCH 50 growth stage [15] to assess their morphophysiological responses. The remaining plants were grown to maturity and harvested, the pods collected, and the grains counted and weighed.

Aqueous Suspension of Boron Nanoparticles
An aqueous suspension was prepared using boron (B 2 O 3 particle size: up to 100 nm; purity: 95%) nanoparticles (US Research Nanomaterials, Inc, Houston, TX, USA) and deionized water at 12.5, 25, and 50 ppm concentrations. Before treatment with nanoparticles, the suspension was dispersed using an ultrasonic bath (Sonerex super ultrasonic bath 80W, Weidinger GmbH, Gernlinden, Germany) for 60 min. The stability of this suspension was evaluated using a particle size meter (Delsa™ Nano Submicron Particle Size, Beckman Coulter Instruments. Corporation, Fullerton, California) and a zeta potential device (Dispersion Technology Inc., Bedford Hills, New York). The pH of the suspension was determined using a pH meter (Hanna instruments, HI5000, Washington, USA).

Research Object
Green pea (Pisum sativum L.) cultivar 'Respect' (Maribo Seed International ApS, Denmark) was used in experiments. It is a medium-early semi-leafless pea variety. Green peas (Pisum sativum L.) were selected as the research object. Peas are the most drought-sensitive members of the legume family and have particular importance in crop rotation. In addition, they fix atmospheric nitrogen in a symbiotic association with Rhizobium bacteria and meet the nitrogen demand of subsequent crops. They are also widely used for both human food and animal feed.

Growth Parameters
Ten plants per treatment were randomly selected (n = 10) for biometric measurements. First, the shoots were separated from the roots, and then the shoot height, root length, fresh weight (FW), and dry weight (DW) were determined. Using electronic scales (Mettler Toledo AG64, Columbus, OH, USA), the FW and DW were measured. DW was determined using a forced-air convection dryer (VENTICELL 222, MBT, Brno, Czech Republic) at 105 ºC. After shoot FW determination, ten matured plants per treatment were floated on deionized water for 24 h, and turgid weights (TW) were measured. Relative water content (RWC) was calculated as described by [16].
The leaf area was measured with an automatic leaf area meter (AT Delta-T Devices, Wallingford, UK) and expressed as cm 2 g −1 . Specific leaf area (SLA) was calculated by dividing the total plant leaf area (n = 10) by shoot DW. The root/shoot ratio was determined as the ratio of root DW to aboveground DW. Pods were collected from each pea plant, and the average number of heads/pods m −2 was counted (A, average number of pods m −2 ). Then, the number of grains in the pods in each variant was calculated, and the average (B, average number of grains per pod) was derived. The weight of 100 grains (C, weight of 100 grains of peas) was calculated. The pea yield was calculated according to the following formula [17]: The pea yield (t ha −1 ) = (A × B × C)/10,000 (2)

Biochemical Analysis
Antioxidant properties of pea leaves were evaluated as the DPPH (2-diphenyl-1picrylhydrazyl), ABTS (2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) diammonium salt, and radical scavenging activities, and the Fe 2+ reducing antioxidant power assay (FRAP). Moreover, the total content of phenolic compounds was determined. Extracts were prepared by grinding 0.3 g of plant leaves with liquid nitrogen and diluting this with 5 mL of 80% methanol. Then, 24 h later, the samples were centrifuged for 10 min at 3000 rpm (Hermle Z300K, Baden-Württemberg, Germany). Cellulose filters were used for extract filtration. The supernatant was used for further analyses. All biochemical analysis was performed in 3 biological replications. Each of the three biological replicates consisted of at least three conjugated plants and was repeated in three analytical replicates.

Non-Enzymatic Antioxidant Activity
The total content of phenolic compounds was determined as gallic acid equivalents. First, a 250 µL aliquot of the sample extract was mixed with 250 µL of 10% (w/v) Folin-Ciocalteu reagent, 500 µL of 1 M Na 2 CO 3 solution, and 2 mL of distilled water [18]. After 20 min incubation in the dark, the absorbance was measured at 765 nm (M501, Spectronic Camspec Ltd., Leeds, UK). The total phenolic compound quantity mg g −1 was calculated from the calibration curve of gallic acid (0.01-0.1 mg mL −1 , R 2 = 0.99).

Enzymatic Antioxidant Activity
The extracts used to determine the enzymatic antioxidant activity in pea leaves were prepared by grinding 0.5 g of fresh sample with liquid nitrogen and diluting within 5 mL extraction buffer (100 mM potassium phosphate buffer, pH 7.8, containing 0.1 mM EDTA). After centrifugation for 10 min at 3000 rpm (Hermle Z300K, Baden-Württemberg, Germany), the supernatant was collected and used for the assays of enzymatic activity. All steps in the preparation of the enzyme extract were carried out at 4 • C.
The dye-binding method and bovine serum albumin as a standard were used to determine soluble proteins. First, 30 µL of enzyme extract was mixed with 1.5 mL of Bradford reagent diluted by 1:5 with DI water. Absorbance was read after 2 min through a spectrophotometer (M501, Spectronic Camspec Ltd., Leeds, UK) at 595 nm [22].
Superoxide dismutase (SOD) activity was estimated by the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) by the enzyme [23]. Here, 3 mL of reaction mixture consisted of 13 mM methionine, 75 µM NBT, 100 mM potassium phosphate buffer (pH 7.8, containing 0.1 mM EDTA), 50 µL enzyme extract, and 13 µM riboflavin. The tubes were kept under 150 µmol m −2 s −1 for 1 min to initiate the reaction and then covered. The absorbance was recorded after 30 min with a spectrophotometer (M501, Spectronic Camspec Ltd., Leeds, UK) at 560 nm, and one unit of enzyme activity was taken as the amount of enzyme that reduced the absorbance reading to 50% in comparison with tubes lacking the enzyme, expressed as unit mg −1 protein min −1 .
Catalase (CAT) activity was measured as the disappearance of H 2 O 2 [24]. First, 100 µL enzyme extract was added to 1.275 mL of 0.1 M phosphate buffer (pH 7.8, containing 0.1 mM EDTA). The reaction was started by adding 125 µL of 30 mM H 2 O 2 (30%, Merck KGaA, Darmstadt, Germany). The decrease in absorbance measured by a spectrophotometer (M501, Spectronic Camspec Ltd., Leeds, UK) at 240 nm was observed for 1 min, and enzyme activity was computed by calculating the amount of H 2 O 2 decomposed (µmol H 2 O 2 mg −1 protein min −1 ).
Ascorbate peroxidase (APX) activity was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm [25]. The 1 mL assay mixture contained 0.1 M potassium phosphate buffer (pH 7.8, containing 0.1 mM EDTA), 0.5 mM ascorbic acid, 0.1 mL enzyme extract, and 0.1 mL of 30 mM H 2 O 2 (30%, Merck KGaA, Darmstadt, Germany) which was added to initiate the reaction. The decrease in absorbance was measured spectrophotometrically (M501, Spectronic Camspec Ltd., Leeds, UK) for 1 min. The extinction coefficient of 2.8 mM −1 cm −1 for reduced ascorbate was used to calculate the enzyme activity, which was expressed as µmol AsA mg −1 protein min −1 .
Glutathione reductase (GR) activity was measured based on the decrease in the absorbance of oxidized glutathione (GSSG) at 340 nm [26]. The reaction mixture contained 0.1 M potassium phosphate buffer (pH 7.8, containing 0.1 mM EDTA), 1 mM GSSG (Merck KGaA, Darmstadt, Germany), 100 µL enzyme extract, and 75 µL 0.1 mM NADPH added last to initiate the reaction. The decrease in absorbance measured by a spectrophotometer (M501, Spectronic Camspec Ltd., Leeds, UK) was recorded every 5 min for 20 min. An absorption coefficient of 6.22 mM −1 cm −1 was used for calculations. GR activity was defined as µmol NADPH mg −1 protein min −1 .

Oxidative Stress Biomarkers
The extracts used to determine the concentration of lipid peroxidation and hydrogen peroxide (H 2 O 2 ) in pea leaves were prepared by grinding 0.1 g of fresh sample with liquid nitrogen and diluting it with 4 mL of 0.1% trichloroacetic acid (TCA). After centrifugation for 10 min at 3000 rpm (Hermle Z300K, Baden-Württemberg, Germany), the supernatant was used for further analyses.
For H 2 O 2 measurements in plant leaves, 500 µL of the supernatant was added to 1 mL of 1 M potassium iodide (KI). The absorbance of the mixture was scanned at 390 nm using a spectrophotometer (M501, Spectronic Camspec Ltd., Leeds, UK). A calibration curve was determined using H 2 O 2 (30%, Merck KGaA, Darmstadt, Germany) as an external standard, with a range of concentrations from 0.6 to 24.3 mM (R 2 = 0.99). The content of H 2 O 2 is expressed as fresh weight (µmol g −1 FW) [28].
The thiobarbituric acid (TBARS) test determines the malondialdehyde (MDA) content in pea leaf samples as the end product of lipid peroxidation. First, 500 µL of the supernatant was added to 1 mL 0.5% (w/v) thiobarbituric acid (TBA, Merck KGaA, Darmstadt, Germany) in 20% trichloroacetic acid (TCA, Merck KGaA, Darmstadt, Germany). The mixture was incubated in boiling water for 30 min. The reaction stopped after the samples had cooled. The samples were centrifuged (Hermle Z300K, Baden-Württemberg, Germany) at 10,000× g for 5 min, and the absorbance of the supernatant was measured at 532 nm using a spectrophotometer (M501, Spectronic Camspec Ltd., Leeds, UK). The value for non-specific absorbance at 600 nm was subtracted [29]. The amount of MDA-TBA complex (red pigment) in leaves was calculated and expressed as nmol g −1 FW: C MDA -concentration of MDA, µM; A 532 , A 600 -absorbance at wavelength; E MDA -MDA extinction coefficient 155 mM −1 cm −1 .

Elemental Composition Analysis
The macro-and microelement quantities in pea leaves, stems, and roots were determined using the microwave digestion technique combined with inductively coupled plasma optical emission spectrometry [30,31]. Complete digestion of dry plant material (0.3 g) was achieved with 8 mL 65% HNO 3 using a microwave digestion system, Multiwave GO (Anton Paar GmbH, Graz, Austria). The digestion program was as follows: (1) 170 • C reached within 3 min, digested for 10 min; (2) 180 • C reached within 10 min, digested for 10 min. Fully digested samples were diluted to 50 mL with deionized water. The elemental profile was analyzed using an ICP-OES spectrometer (Spectro Genesis, SPECTRO Analytical Instruments, Kleve, Germany). The operating conditions employed for ICP-OES determination were 1300 W RF power, 12 L min −1 plasma flow, 1 L min −1 auxiliary flow, 0.8 L min −1 nebulizer flow, and 1 mL min −1 sample uptake rate. The analytical wavelengths chosen were P I 213. 618  The calibration standards were prepared by diluting a stock multi-elemental standard solution (1000 mg L −1 ) in 6.5% (v/v) nitric acid and by diluting stock phosphorus and standard sulfur solutions (1000 mg L −1 ) in deionized water. The calibration curves for all the studied elements were in the range of 0.01-400 mg L −1 . The levels of macro-and microelements in the dry weight of pea are presented.

Statistical Analysis
All the values were presented as mean ± standard deviation. Data were analyzed using the Analysis of Variance (ANOVA) test followed by Tukey's HSD at p ≤ 0.05 to identify significant differences (XLStat software, Addinsoft, Paris, Ile-de-France, France, 2022). Differences were analyzed between variants when peas were grown under normal conditions and separately between peas grown under drought conditions. The micro-and macroelement analysis results were also compared between controls, i.e., peas grown in a drought and not treated with NPs (SM 30%) and treated with NPs, and between control peas grown under normal conditions (SM 80%) not treated with NPs and treated with NPs.

Boron Nanoparticles' Impact on Morphological Parameters
Pea height increased by 14 and 27% when watered and by 28 and 19% when sprayed with 12.5 and 50 ppm B 2 O 3 NP suspension under sufficient substrate moisture (Table 1, 80% SM). Furthermore, a positive effect of B 2 O 3 NPs was found in the pea plant's leaf area and RWC. At the same time, a decrease of 15% in SLA was observed after spraying with 12.5 ppm solution. The root-to-shoot ratio statistically reliably increased after watering plants at 12.5 ppm by 21%, 25 ppm-by 36%, and 50 ppm-by 68%. In addition, an increase in the root-to-shoot ratio by 68% (12.5 ppm), 18% (25 ppm), and 34% (50 ppm) was observed when plants were sprayed. The results also showed that B 2 O 3 NPs positively affected the number of nodules on plant roots by increasing their amount by up to 5.6 times when plants were watered and up to 3.4 times when plants were sprayed. The results showed that pea irrigation with 50 ppm B 2 O 3 NPs had a significant positive effect on yield, while foliar treatment increased the pea yield for suspensions containing 12.5 and 25 ppm B 2 O 3 NPs.

Effects on Oxidative Stress Biomarkers
The results show that exposure to B 2 O 3 NPs through the roots increased the amount of H 2 O 2 in plants, regardless of the concentration, when peas were grown under sufficient substrate moisture ( Figure 1A, 80% SM). When plants were sprayed, a statistically reliable 65% increase in H 2 O 2 content was found at 12.5 ppm B 2 O 3 NPs. A significant decrease in the MDA concentration ( Figure 1B, 80% SM)

Effects on Oxidative Stress Biomarkers
The results show that exposure to B2O3 NPs through the roots increased the amount of H2O2 in plants, regardless of the concentration, when peas were grown under sufficient substrate moisture ( Figure 1A, 80% SM). When plants were sprayed, a statistically reliable 65% increase in H2O2 content was found at 12.5 ppm B2O3 NPs. A significant decrease in the MDA concentration ( Figure 1B, 80% SM) was also found in pea leaves as plants were watered or sprayed with a solution containing any concentration of B2O3 NPs.
The significant inhibition of H2O2 and MDA was found as their concentration decreased after plants' exposure to drought and B2O3 NPs ( Figure 1A, B 30% SM). The amount of H2O2 decreased by 18, 24, and 45% after spraying the plants with 12.5, 25, and 50 ppm, and by 22, 37, and 9% after watering. A reduction in the MDA content by 22, 13, and 17% was found after pea irrigation with 12.5, 25, and 50 ppm suspensions of B2O3 NPs and after foliar application by 20, 25, and 22%.

Effects on Non-Enzymatic Antioxidants
It was found that at 80% substrate moisture, watering and spraying with B2O3 NPs reduced the TPC in pea leaves by up to 30% (Figure 2A, 80% SM). B2O3 NP treatment did The significant inhibition of H 2 O 2 and MDA was found as their concentration decreased after plants' exposure to drought and B 2 O 3 NPs ( Figure 1A, B 30% SM). The amount of H 2 O 2 decreased by 18, 24, and 45% after spraying the plants with 12.5, 25, and 50 ppm, and by 22, 37, and 9% after watering. A reduction in the MDA content by 22, 13, and 17% was found after pea irrigation with 12.5, 25, and 50 ppm suspensions of B 2 O 3 NPs and after foliar application by 20, 25, and 22%.

Effects on Non-Enzymatic Antioxidants
It was found that at 80% substrate moisture, watering and spraying with B 2 O 3 NPs reduced the TPC in pea leaves by up to 30% (Figure 2A, 80% SM). B 2 O 3 NP treatment did not affect ABTS free radical scavenging activity ( Figure 2C, 80% SM). However, it was determined that after spraying peas with 25 and 50 ppm suspensions, the DPPH free radical scavenging activity increased by 25 and 24% ( Figure 2B, 80% SM). Furthermore, concentrations of B 2 O 3 NP suspensions of 12.5, 25, and 50 ppm increased the FRAP antioxidant power ( Figure 2D, 80% SM), as plants were watered or sprayed.  The results showed that spraying drought-affected peas with 12.5, 25, and 50 ppm B 2 O 3 NP suspensions increased the TPC content to 18%, while watering with 12.5 ppm significantly reduced it (Figure 2A, 30% SM). ABTS free radical scavenging activity showed sensitivity to the impact of B 2 O 3 NPs ( Figure 2C, 30% SM); it increased to 73% after watering and 96% after spraying compared to drought-affected plants without NP exposure. Similar results were found for FRAP antioxidant power in peas ( Figure 2D, 30% SM). The exposure to drought and B 2 O 3 NP 12.5 and 25 ppm suspensions through the roots exerted a slight impact (20%) on DPPH free radical scavenging activity ( Figure 2B 30% SM). In addition, spraying with 12.5, 25, and 50 ppm B 2 O 3 NP suspensions induced DPPH free radical scavenging activity by 35, 24, and 25%, respectively.

Effects on Enzymatic Antioxidants
NPs induced the activity of CAT, APX, SOD, and GPX in pea leaves when they were grown in 80% SM ( Figure 3A,B,D,E). APX activity increased particularly strongly after watering plants with B 2 O 3 NP suspensions, while a slightly weaker effect was caused by spraying. CAT activity increased up to two times when plants were watered with suspensions of B 2 O 3 NPs. When peas were sprayed, the CAT activity increased by 1.3, 1.8, and 2 times when the concentration was 12.5, 25, and 50 ppm. SOD activity was induced by up to 41% by exposure to B 2 O 3 NPs through roots, and foliar treatment activated the enzyme by up to 46%. GPX activity was distinguished because lower concentrations of 12.5 and 25 ppm had a more substantial positive effect during watering, while higher concentrations of 25 and 50 ppm increased the activity more strongly during spraying. The B 2 O 3 NP suspension reduced the GR activity ( Figure 3C, 80% SM) when suspensions with concentrations of 12.5 and 50 ppm were used for plant watering or spraying.
A substantial decrease in GR activity was caused by drought and B 2 O 3 NP exposure, with a 55% reduction after irrigation and a 45% reduction after spraying ( Figure 3C, 30% SM). Moreover, an adverse effect was found on SOD activity ( Figure 3D, 30% SM); after peas' irrigation with 25 and 50 ppm solutions of B 2 O 3 NPs, a 36% increase in SOD activity was determined after using the 12.5 ppm B 2 O 3 NP suspension. Furthermore, SOD activity was induced by up to 51% when drought-affected peas were sprayed with the B 2 O 3 NP solution. The strong effect of B 2 O 3 NPs on the APX activity ( Figure 3B, 30% SM) remained in pea leaves as plants were grown in drought conditions. After watering peas with B 2 O 3 NP suspensions, APX activation occurred by up to 1.4 times after spraying up to eight times. Additionally, GPX activity in drought-affected peas ( Figure 3E, 30% SM) was increased by up to 91% after foliar exposure with all B 2 O 3 NP concentrations. CAT activity ( Figure 3A, 30% SM) was strongly activated by the watering or spraying of plants with B 2 O 3 NP solutions of any concentration.

Comparison and Summary of Results
As can be seen in the heat map (Table 2), in peas grown with normal substrate moisture and watered or sprayed with B 2 O 3 NPs, nodulation, FRAP, hydrogen peroxide formation, GPX, APX, and CAT were most strongly induced, but ABTS antioxidant capacity, TPC and MDA content, and GR activity were reduced. Strong nodulation; increased ABTS antioxidant capacity, FRAP antioxidant power, GPX, APX, and CAT activity; and significantly decreased H 2 O 2 and MDA levels were observed in peas grown under drought conditions with B application. Notably, nodule formation increased with an increasing concentration, but the activity and content of most antioxidants decreased, although they were still higher than in plants grown under drought conditions but without B 2 O 3 NPs. The yield of peas grown under drought conditions was higher with the corresponding increase in antioxidant content and activity, which means that as the concentration of B 2 O 3 NPs decreased, the yield increased, and it even increased by up to 19% compared to B 2 O 3 -untreated plants.

Discussion
In our studies, the zeta potential of the aqueous suspension of B 2 O 3 NPs was −28.54 mV (Table 1). However, other researchers found that an aqueous suspension of B 2 O 3 NPs with 0.2% Triton X-100 had a value −30.3 mV [32]. Such zeta potential values indicate that the solutions are stable and anionic. In addition, the PDI of this suspension was 0.23, while other scientists have found a value of 0.4 [32], indicating that the suspensions are monodisperse.
To better understand the effects of B on plants, it is important to determine the form and mechanism with which B enters the plant naturally. Around 96% of boron exists mainly as boric acid (H 3 BO 3 ) and a small amount as borate anion [B(OH) 4 − ] at a neutral pH of 5.5-7.5 [33]. Such forms of B can readily diffuse through roots or be transported by major intrinsic protein (MIP) channels or BOR transporters depending on the plant species [34].
The study shows that the number of nodules formed on pea roots (Table 2) increases strongly after exposure to B 2 O 3 NPs in both regular and deficient substrate moisture conditions. Such an effect could be explained by the fact that once B enters the plant, it is transported in the xylem, and approximately 90% is incorporated into plant cell walls [35]. B is the main element in the formation of esters with rhamnogalacturonan II (RGII). This borate ester is required to maintain normal cell wall functions and structures [36]. Under normal conditions, when there is sufficient B in the soil, RGII glycoproteins are also formed in the plasma membranes of pea root nodules and root cells. However, RGII glycoproteins are not synthesized, and their absence destabilizes the plasma membrane and nodule formation in B deficiency [37]. Moreover, B, as a component of glycoproteins, is essential for differentiating nodule bacteria into a nitrogen-fixing form [38]. Increased nitrogen fixation in the pea plant increases its resistance, resulting in increased antioxidant production, as seen in our results ( Table 2).
It is worth mentioning that our study expands the knowledge about the effect of B 2 O 3 NPs on the antioxidant systems of plants. It should be emphasized that B 2 O 3 NPs effectively protect plants from drought stress by stimulating non-enzymatic FRAP antioxidant power and ABTS free radical scavenging activity and APX, GPX, and CAT enzymatic antioxidants. Moreover, B NPs can also influence the shoot height, flower number, and B, N, and K accumulation in soybeans [4]. Furthermore, algae treated with B NPs showed higher Chl content and MDA and H 2 O 2 concentrations, and more active SOD and CAT enzymes [39]. In addition, B deficiency affected >70% of the analyzed genes [7]. Most were upregulated, but some genes critical for nodule development and function were downregulated.
NPs can help to maintain the pea yield even under adverse environmental conditions. Their effect depends on the concentration and method of exposure. B 2 O 3 NPs have a positive effect on the antioxidant system of peas, reducing the amount of oxidative stress biomarkers, which positively affects growth indicators, but more detailed studies are needed to evaluate their overall effects on different ecosystems. These findings contribute to the application of nanoparticles in agronomy but we do not recommend their use in practice until their effects on different ecosystems are studied.

Conclusions
Spraying with B 2 O 3 nanoparticles at 12.5 ppm most effectively stimulated phenol accumulation, antioxidant capacity, ascorbate peroxidase, superoxide dismutase, and guaiacol peroxidase enzyme activity in pea leaves exposed to drought. In addition, B 2 O 3 nanoparticles reduced the amount of hydrogen peroxide and malondialdehyde in pea plants grown on a substrate with insufficient moisture. The most substantial positive effect was found on peas affected by drought after spraying them with 12.5 ppm of B 2 O 3 nanoparticles. These findings contribute to the application of nanoparticles in agronomy, but we do not recommend their use in practice until their effects on different ecosystems are studied.